1. Field of the Invention
The invention relates to the field of methods and apparatus for using a fiber-based probe and/or fiber-based endoscope for coherent anti-Stokes Raman scattering (CARS) imaging of a sample.
2. Description of the Prior Art
Nonlinear optical microscopy is a powerful imaging approach with applications found in areas as diverse as biology, medicine, and physics. Nonlinear optical microscopy includes the techniques of two-photon excited fluorescence (TPEF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. Coherent anti-Stokes Raman spectroscopy, also called coherent anti-Stokes Raman scattering spectroscopy (CARS), is a form of spectroscopy used primarily in chemistry, physics and related fields. It is sensitive to the same vibrational signatures of molecules as seen in Raman spectroscopy, typically the nuclear vibrations of chemical bonds. Unlike Raman spectroscopy, CARS employs multiple photons to address the molecular vibrations, and produces a signal in which the emitted waves are coherent with one another. As a result, CARS is orders of magnitude stronger than spontaneous Raman emission. CARS is a third-order nonlinear optical process involving three laser beams: a pump beam of frequency ωp, a Stokes beam of frequency ωS and a probe beam at frequency ζpr. These beams interact with the sample and generate a coherent optical signal at the anti-Stokes frequency (ωp−ωS+ωpr). The latter is resonantly enhanced when the frequency difference between the pump and the Stokes beams (ωp−ωS) coincides with the frequency of a Raman resonance, which is the basis of the technique's intrinsic vibrational contrast mechanism.
This imaging approach as gained enormous popularity in biomedical imaging of tissues in vivo, because it provides high resolution images at fast imaging acquisition rates. Moreover, nonlinear optical microscopy does not necessitate labels as it derives its contrast from endogenous structures in the tissue. Typical applications include skin and superficial tissue imaging, often in an in vitro setting. While the potential of nonlinear microscopy for in vivo imaging is high, the actual implementation of the combined nonlinear imaging approach in the clinic requires suitable fiber-delivered probes that are currently unavailable. Several kinds of fiber-delivered and endoscopic probes have already been developed, most notably probes for optical coherence tomography (OCT), second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). However, a suitable fiber-delivered probe that enables CARS imaging in addition to SHG and TPEF imaging is currently lacking.
From a design point of view, the development of a fiber-delivered or endoscopic probe that supports CARS imaging imposes additional challenges to existing probe designs. The reason for this is that unlike SHG and TPEF, CARS incorporates two laser beams, called ‘Pump’ and ‘Stokes’, that both need to be guided and focused onto the sample. Finding suitable fibers that support delivery of both laser beams without compromising the image quality has been one of the design challenges.
Another design challenge is to collect the signal, emitted at the anti-Stokes wavelength, and guide that radiation through a fiber toward a photodetector. US Pat. Pub. No. 2007/0088219 “System and Method for Coherent Anti-stokes Raman Scattering Endoscopy” discloses a system and method for fiber based CARS endoscopy. This document discloses a system for guiding both the Stokes and pump wave in one optical fiber and receive the anti-Stokes signal emitted from the sample with the same fiber or different fiber. However, it is found that strong four-wave-mixing (FWM)/CARS contribution at the anti-Stokes frequency is generated in the delivery fiber under typical CARS excitation conditions. This fiber-generated FWM/CARS component forms a large background that is typically overwhelming the signal generated in the sample, and severely complicates the interpretation of the image unless removed. This major problem is not discussed in US Pat. Pub. No. 2007/0088219, and significantly limits the application of the device proposed therein. The mixing the anti-Stokes radiation generated in the delivery fiber with the CARS signal generated from the sample forms a major limitation of the existing technology. In order to get the actual CARS image of the sample, the anti-Stokes frequency generated in the delivery fiber must be filtered out before it is focused onto the sample.